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We have determined the three-dimensional structure of bacteriophage SPP1 portal protein (gp6) using electron microscopy at liquid-helium temperatures and angular reconstitution. The 13-fold symmetric gp6 oligomer is a turbine-shaped structure with three distinct regions: a conical stem with a central channel; the turbine wings region; and a fringe of small 'tentacles' at the end of the channel exposed to the viral head interior. The tentacle region appears flexible and may be associated with a particular function — sensing when the correct amount of DNA has been packaged. The three-dimensional structure of the gp6 SizA mutant, which packages a smaller chromosome, reveals significant differences in that region.

This tutorial paper is the mannscript of a lecture given in the Harnack-Haus in Berlin during the antumn school 1998 on Metal Clusters. The aim of this lecture was to give some hints for quantitative electron microscopy. The interpretation of electron microscopic images depends on the mode of contrast. In particular the phase contrast is described and how it affects the images.

Samples of 80 S ribosomes from rabbit reticulocytes were subjected to electron cryomicroscopy combined with angular reconstitution. A three-dimensional reconstruction at 21 A resolution was obtained, which was compared with the corresponding (previously published) reconstruction of Escherichia coli 70 S ribosomes carrying tRNAs at the A and P sites. In the region of the intersubunit cavity, the principal features observed in the 70 S ribosome (such as the L1 protuberance, the central protuberance and A site finger in the large subunit) could all be clearly identified in the 80 S particle. On the other hand, significant additional features were observed in the 80 S ribosomes on the solvent sides and lower regions of both subunits. In the case of the small (40 S) subunit, the most prominent additions are two extensions at the base of the particle. By comparing the secondary structure of the rabbit 18 S rRNA with our model for the three-dimensional arrangement of E. coli 16 S rRNA, these two extensions could be correlated with the rabbit expansion segments (each totalling ca 170 bases) in the regions of helix 21, and of helices 8, 9 and 44, respectively. A similar comparison of the secondary structures of mammalian 28 S rRNA and E. coli 23 S rRNA, combined with preliminary modelling studies on the 23 S rRNA within the 50 S subunit, enabled the additional features in the 60 S subunit to be sub-divided into five groups. The first (corresponding to a total of ca 335 extra bases in helices 45, 98 and 101) is located on the solvent side of the 60 S subunit, close to the L7/L12 area. The second (820 bases in helices 25 and 38) is centrally placed on the solvent side of the subunit, whereas the third group (totaling 225 bases in helices 18/19, 27/29, 52 and 54) lies towards the L1 side of the subunit. The fourth feature (80 bases in helices 78 and 79) lies within or close to the L1 protuberance itself, and the fifth (560 bases in helix 63) is located underneath the L1 protuberance on the interface side of the 60 S subunit.

Background: The ribosome is central to protein synthesis in all living organisms. Single-particle electron cryomicroscopy has recently led to the determination of three-dimensional structures of bacterial ribosomes to ∼20 a, which have since revolutionised our understanding of ribosomal function. The structure we present here of the 80S rat liver ribosome leads the way to similar progress for mammalian ribosomes. Results: Among the new details revealed by our 25 a structure of the 80S rat liver ribosome are channels within the subunits, a large ‘flat ribosomal surface' (FRS) on the outer surface of the large subunit and structural extensions of the mammalian compared to the bacterial ribosome. The main large subunit channel in both the bacterial and the mammalian species starts at the peptidyl transferase centre, below the central protuberance, and ends in the FRS, at the lower back of the large subunit. Structurally, the channels of both species can be directly superimposed. Conclusions: The mammalian structural extensions – none of which trespass the FRS – can be interpreted in terms of rRNA inserts and additional protein content over that of bacterial ribosomes. The main large subunit channel, which ends at the FRS, is the best candidate for the exit channel for proteins targeted for the endoplasmic reticulum.

Lattice images of dislocations, growth boundaries and surface structure, of the n-paraffin series have been obtained by high resolution electron microscopy. The crystals were grown so that the chai...

A three-dimensional reconstruction of keyhole limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to ∼45 A resolution, the new 15 A resolution reconstruction is very similar to the earlier reconstructions of gastropodan hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the decameric cephalopod hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the “functional units” within the dimeric “morphological units” of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical hemocyanin. Keyhole limpet hemocyanin is used clinically as an immunostimulant. The very strong immune reaction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components.

Electron microscopy of biological structural research has made significant advances. These advances were the stimulus to develop an electron microscope containing most of the desirable features and methods for high resolution cryo-electron microscopy. The new instrument is a modified commercial Philips CM 20 FEG electron microscope equipped with a helium-cooled cryoobjective from Siemens. This paper presents the essential features and research possibilities of this new microscope named “SOPHIE” (acronym for S uperconducting O bjective in a PHI -lips E lectron microscope).

A series of diffraction patterns from two-dimensional protein crystals of bacteriorhodopsin (purple membrane) at different temperatures (294 K, 98 K and 4 K) were recorded as the diffraction spots faded due to radiation damage. The patterns were then computationally evaluated in order to obtain a quantitative measurement of the structural preservation while irradiating the specimen. To provide statistically significant results, diffraction spots corresponding to spacings of 3A(1200 spots) and 7A(600 spots) were measured. A substantial increase of the lifetime of high resolution spots was found using liquid nitrogen as a coolant, whereas further structural preservation at liquid helium temperature was significant but smaller. It appears likely therefore that high resolution images are accessible even at liquid nitrogen temperature. Mechanical stability and the absence of thermal specimen drift are certainly of equivalent importance for successful high resolution imaging.

A high resolution projection at 2.6 A of deoxycholate-treated purple membrane using only images has been obtained with a 200 keV FEG microscope operated at liquid helium temperature. Examination of this high quality map has allowed the following conclusions to be made: Comparison between the internal structure of the trimers of the native and the deoxycholate-treated crystal forms shows that almost every detail of the structure at high resolution is identical. The cell dimension change from 62.4 A to 57.9 A is accompanied by a loss of about half the normal lipids and a 2 degrees anticlockwise rotation of the trimer as a rigid body. Three of the lipids per bacteriorhodopsin molecule remain in identical positions relative to the trimer. In addition, from the projection map together with a packing analysis using the atomic model for bacteriorhodopsin, space for three further lipids has been identified making a total of six lipids per bacteriorhodopsin molecule in this crystal form. Finally, the surprisingly small rotation of the trimer between the two crystal forms with completely different Van der Waals contacts suggest that the crystals are held together by strong, long-range electrostatic interactions.

Using cryo-microscopy at liquid-helium temperature we have determined the projection structure of the surface layer of the archaebacterium Sulfolobus shibatae embedded in aurothioglucose to a resolution of 0.85 nm. Image analysis was performed in real space and incorporated a classification of unit cells, thus taking into account the frequent occurrence of twin boundaries. In an attempt to identify resolution-limiting factors we have performed a distortion analysis quantifying the main distortion parameters.

Based on experience gained with the helium-cooled cryomicroscope “Suleika” [I. Dietrich et al., Ultramicroscopy 2 (1977) 241], the requirements for a microscope that also satisfies coherency conditions are developed and discussed.

This work examines the effect of temperature on the rate of damage caused by the electron beam in the microscope. Oriented single-crystal films of aromatic organic compounds have been used as specimens between 4 and 293 K. It is shown that the rate of damage is a function of the thickness and specimen temperature for any one compound, and that the rates of damage to the same specimen are exponentially related to reciprocal temperature. The Arrhenius plots reveal two activation energies for the degradation reactions with the higher activation energy ceasing at approximately 80 K. It would be expected that significant advantages in specimen lifetime would be obtained in specimens examined below this temperature.

In recent years, electron crystallography has been a very successful means of determining the high-resolution structure of many proteins, such as bacteriorhodopsin, the light-harvesting complex, porins (PhoE and OmpF), protein extracted from the ootheca of the praying mantis, archaebacterial surface layers, acetylcholine receptor, Ca 2+ - ATPase and gp32 ∗ l crystals. This success was based on advanced low-dose cryoelectron microscopy and a highly sophisticated digital image-processing methodology which takes into account the radiation damage caused by electron exposure. Particularly successful has been the structure determination of the purple membrane where, by combining biochemical information with the electron-microscopical density maps, the amino acid chains of bacteriorhodopshin have been localized with high precision.

Crystal structure analyses of polymethylene compounds via direct phasing methods are notoriously difficult because of the dominance of the diffraction intensity by the scattering from polymethylene lattice. However, by combination of high resolution low-dose images with conventional direct phasing via structure-invariant relationships, one can assign phase values to the most important regions of the diffraction pattern (i.e. “lamellar” and “polyethylene” reflections, respectively). As demonstrated here with experimental data from the epitaxially crystallized paraffin, n -hexatriacontane, the phased observed structure factor magnitudes produce an electrostatic-potential map with atomic positions identical to the previously published X-ray crystal structure.

The mRNA codon in the ribosomal A-site is recognized by aminoacyl-tRNA (aa-tRNA) in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here we report the 13 A resolution three-dimensional reconstruction determined by cryo-electron microscopy of the kirromycin-stalled codon-recognition complex. The structure of the ternary complex is distorted by binding of the tRNA anticodon arm in the decoding center. The aa-tRNA interacts with 16S rRNA, helix 69 of 23S rRNA and proteins S12 and L11, while the sarcin-ricin loop of 23S rRNA contacts domain 1 of EF-Tu near the nucleotide-binding pocket. These results provide a detailed snapshot view of an important functional state of the ribosome and suggest mechanisms of decoding and GTPase activation.

We have determined the three-dimensional structure of bacteriophage SPP1 portal protein (gp6) using electron microscopy at liquid-helium temperatures and angular reconstitution. The 13-fold symmetric gp6 oligomer is a turbine-shaped structure with three distinct regions: a conical stem with a central channel; the turbine wings region; and a fringe of small 'tentacles' at the end of the channel exposed to the viral head interior. The tentacle region appears flexible and may be associated with a particular function - sensing when the correct amount of DNA has been packaged. The three-dimensional structure of the gp6 SizA mutant, which packages a smaller chromosome, reveals significant differences in that region.

Ordering of a system of particles into its thermodynamically stable state usually proceeds by thermally activated mass transport of its constituents. Particularly at low temperature, the activation barrier often hinders equilibration--this is what prevents a glass from crystallizing and a pile of sand from flattening under gravity. But if the driving force for mass transport (that is, the excess energy of the system) is increased, the activation barrier can be overcome and structural changes are initiated. Here we report the reordering of radiation-damaged protein crystals under conditions where transport is initiated by stress rather than by thermal activation. After accumulating a certain density of radiation-induced defects during observation by transmission electron microscopy, the distorted crystal recrystallizes. The reordering is induced by stress caused by the defects at temperatures that are low enough to suppress diffusive mass transport. We propose that this defect-induced reordering might be a general phenomenon.

A three-dimensional reconstruction of keyhole limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to approximately 45 A resolution, the new 15 A resolution reconstruction is very similar to the earlier reconstructions of gastropodan hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the decameric cephalopod hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the "functional units" within the dimeric "morphological units" of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical hemocyanin. Keyhole limpet hemocyanin is used clinically as an immunostimulant. The very strong immune reaction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components.

A three-dimensional map of bacteriorhodopsin has been obtained, at near-atomic resolution, by collecting and analysing electron diffraction patterns and electron micrographs from crystals of bacteriorhodopsin preserved at very low temperatures. The map shows a resolution of 3.5 degrees in a direction parallel to the plane of the membrane, but poorer resolution perpendicular. It shows many features well resolved from the main density of the seven alpha-helices, which we interpret as the bulky sidechains of tyrosine, phenylalanine and tryptophan, as well as a very dense feature, which is the beta-ionone ring of the retinal chromophore. Using these bulky side chains as starting points and taking account of bulges of density for the smaller side chains such as leucine, we built an atomic model for the residues between 8 and 225. There are 21 amino acids from all 7 helices surrounding the retinal and 26 amino acids contributed by 5 helices that form the proton channel. Ten of the amino acids in the middle of the proton channel are also part of the retinal-binding site. The model provides a useful basis for considering the mechanism of proton pumping and in the interpretation of other experimental data. In particular, the model suggests that the pK changes in the Schiff base must act as the means by which light energy is converted to proton pumping through the channel. Asp-96 is on the pathway from the cytoplasm to the Schiff base and asp-85 on the pathway from the Schiff base to the extracellular surface. The experimental map and the building of the model of the structure will be described, as well as our interpretation of the structural basis of the mechanism.

Cryoelectron microscopy is now proven to be an advantageous tool for structure research of protein crystals. Using a helium-cooled superconducting electron microscope, the following protein crystals have been imaged with high resolution in one projection: crotoxin complex 3.5 Â, purple membrane 2.8 Â, matrix porin OmpF 3.5 Â, surface protein of sulfolobus spec. B12 10 Â. Fine-structure details within the unit cell were resolved, although even at 4.5 K specimen temperature the crystal still suffers considerable radiation damage. While the tolerable dose is only 20 e/Â2, for atomic resolution a dose at least 100 times higher is needed. The ladder to these successes had two steps: First, the image was taken in a special way with a minimum of pre-exposure. Second, the low-dose images were evaluated by a sophisticated image-processing procedure, the main part of which is the cross-correlation averaging. The high resolution structure is in all cases revealed by averaging thousands of low-dose-imaged unit cells.

Low-dose, high-resolution electron microscopy combined with conventional direct-phasing methods based on the estimates of triplet-structure invariants are used to determine phase values for all observed electron-diffraction-structure factor magnitudes from epitaxially oriented multilamellar paracrystals of the phosphospholipid 1,2-dihexadecyl-sn-glycerophosphoethanolamine. The reverse Fourier transform of these phase-structure factors is a one-dimensional electrostatic potential map that strongly resembles the electron-density maps calculated from similar x-ray-diffraction data. Determination of the phase values for the electron-diffraction data with structure invariants alone is nearly as successful as the combined use of two separate methods, assigning values to 13 of the 16 reflections--i.e., the electrostatic potential map closely resembles the one calculated with all data.

The light-driven proton pump bacteriorhodopsin occurs naturally as two-dimensional crystals. A three-dimensional density map of the structure, at near-atomic resolution, has been obtained by studying the crystals using electron cryo-microscopy to obtain electron diffraction patterns and high-resolution micrographs. New methods were developed for analysing micrographs from tilted specimens, incorporating methods previously developed for untilted specimens that enable large areas to be analysed and corrected for distortions. Data from 72 images, from both tilted and untilted specimens, were analysed to produce the phases of 2700 independent Fourier components of the structure. The amplitudes of these components were accurately measured from 150 diffraction patterns. Together, these data represent about half of the full three-dimensional transform to 3.5 A. The map of the structure has a resolution of 3.5 A in a direction parallel to the membrane plane but lower than this in the perpendicular direction. It shows many features in the density that are resolved from the main density of the seven alpha-helices. We interpret these features as the bulky aromatic side-chains of phenylalanine, tyrosine and tryptophan residues. There is also a very dense feature, which is the beta-ionone ring of the retinal chromophore. Using these bulky side-chains as guide points and taking account of bulges in the helices that indicate smaller side-chains such as leucine, a complete atomic model for bacteriorhodopsin between amino acid residues 8 and 225 has been built. There are 21 amino acid residues, contributed by all seven helices, surrounding the retinal and 26 residues, contributed by five helices, forming the proton pathway or channel. Ten of the amino acid residues in the middle of the proton channel are also part of the retinal binding site. The model also provides a useful basis for consideration of the mechanism of proton pumping and allows a consistent interpretation of a great deal of other experimental data. In particular, the structure suggests that pK changes in the Schiff base must act as the means by which light energy is converted into proton pumping pressure in the channel. Asp96 is on the pathway from the cytoplasm to the Schiff base and Asp85 is on the pathway from the Schiff base to the extracellular surface.

Electron cryo-microscopy of individual non-crystallized macromolecules (“single particles”) is a very rapid technique for probing the three-dimensional (“3D”) structure of biological macromolecules. By exploiting the different orientations of the macromolecules in the embedding medium, one may extract 3D information from the data without ever collecting tilt series in the microscope. The angular reconstitution approach, designed for this purpose, was recently extended with a number of refinements which allow its use as a routine technique for finding 3D structures of macromolecules with arbitrary pointgroup symmetry, from entirely asymmetric ribosomes to viruses with icosahedral symmetry.The specimen preparation technique associated with the angular reconstitution approach is simple and fast since crystallization experiments are avoided altogether. The vitreous-ice embedding specimen preparation technique remains one of our favorite specimen preparation techniques. We are, however, currently experimenting with specimens embedded in glucose and ammonium molybdate or other heavy-metal salts. Collecting good micrographs can also be quite straightforward since the images are taken from untilted specimens, while exposing each image area only once.

High-resolution cryo-electron microscopy yielded remarkable results in biological molecular structure research. These successes encouraged to proceed in developing even better instruments and procedures. In our laboratory we built the cryo-electron microscope "SOPHIE", a modified Philips CM 20 FEG. SOPHIE stands as an acronym for Superconducting Objective in a PHIlips Electronmicroscope. The main features of this microscope are the following: 1.200kV acceleration voltage2.Schottky field emitter3.Helium cooled superconducting objective The Helium-cooled cryo-objective from the former microscope SULEIKA was adapted into the column of the original Philips CM20 FEG [Fig. 1], This new SOPHIE microscope, thus, combines two important features: a modern computer controlled electron microscope with a high coherent beam source and the proven advantages of the superconducting cryo-objective. The advantage of the field emission gun at 200 kV over a thermionic cathode at 100 kV can be best visualized while comparing the respective phase-contrast-transfer functions at Scherzer focus [Fig.2]. Moreover, SOPHIE is equipped with the small spot scanning procedure.

Functionality and efficiency of traffic telematics systems depend crucially on the available data base. In our paper we argue that systems based on floating car data (FCD) display – in principle – self-consistency with respect to data generation and usage. Ensuing practical problems of FCD-based telematics systems are discussed. When designing a telematics system for the logistics of commercial traffic, business and operational aspects have to be taken into account. The corresponding systems design is discussed with reference to a tool developed at gedas telematics, the telematics provider in the Volkswagen Group.

The task in investigating the structure of crystals is to measure the Fourier coefficients in amplitude and phase. Electron crystallography, i.e. electron diffraction combined with electron-microscopical imaging, is a straigthforward method, because there is a priori no “phase problem”. But radiation damage during electron exposure was for a long time an absolutely unbridgeable chasm on the way to high-resolution structure research of two-dimensional protein crystals by electron microscopy. Now, using cryo-electron microscopy and computer image processing, it has been proven possible to overcome this difficulty and achieve high resolution.Here we present a rough description of the recording procedure. We used a helium-cooled superconducting objective lens with 4.5 K specimen temperature, but the procedure should also be applicable at higher specimen temperatures. The radiation damage is reduced at low specimen temperature, but unfortunately there still remains a remarkable deterioration of the crystal with increasing dose, which means that minimum exposure is advisable.

The thermoacidophilic archaebacterium Sulfolobus spec. B12 , which is closely related to Sulfolobus solfataricus , possesses a regularly arrayed surface protein (S-layer), which is linked to the plasma membrane via spacer elements spanning a distinct interspace of approximately 18 nm. The S-layer has p3-Symmetry and a lattice constant of 21 nm; three-dimensional reconstructions of negatively stained fragments yield a layer thickness of approximately 6-7 nm.For analysing the molecular architecture of Sulfolobus surface protein in greater detail we use aurothioglucose(ATG)-embedding for specimen preparation. Like glucose, ATG, is supposed to mimic the effect of water, but has the advantage of being less volatile. ATG has advantages over glucose when working with specimens composed exclusively of protein because of its higher density of 2.92 g cm-3. Because of its high radiation sensitivity electromicrographs has to be recorded under strict low-dose conditions. We have recorded electromicrographs with a liquid helium-cooled superconducting electron microscope (the socalled SULEIKA at the Fritz-Haber-lnstitut) with a specimen temperature of 4.5 K and with a maximum dose of 2000 e nm-2 avoiding any pre-irradiation of the specimen.

The light driven proton pump bacteriorhodopsin (bR) occurs naturally as two-dimensional crystals. A three-dimensional density map of the structure, at near atomic resolution, has been obtained by studying the crystals using electron cryo-microscopy to obtain diffraction patterns and high resolution micrographs (1).New methods have been developed for analysing micrographs from tilted specimens, incorporating the methods previously developed for untilted specimens that enable large areas to be analysed and corrected for distortions. Data from 72 images, from both tilted and untilted specimens, have been analysed to produce the phases of 2700 independent Fourier components of the structure. The amplitudes of these components have been accurately measured from 150 diffraction patterns. Together, these data represent about half of the full three-dimensional transform to 3.5 Å. The distribution of the data which is included in the map is shown in fig. 1. For specimen tilts up to around 20° the data is essentially complete. For higher tilts the data is more sparsely sampled, and is at present about half complete.

Many of the most interesting specimens are so severely damaged during the electron microscopical imaging that no useful image emerges from the electron noise. One method of reducing this radiation damage is to cool the specimen, and if this helps it seems reasonable to cool it down as far as possible. This goal is reached most straightforwardly by using the Siemens helium-cooled superconducting objective lens designed by I. Dietrich and co-workers.

The process of electron beam irradiation of orthorhombic monolamellar microcrystals of the n-paraffin n-tetratetracontane cooled < 15K is monitored by changes in the crystal structure to give a much clearer view of the beam damage process than is afforded by plots of diffraction intensity variation. The observed structural change in the chains, observed in the projection down the chain axes as an increasingly circular cross-section, is consistent with previous structural analyses of structural damage in an orthogonal view and also other observations of conformational flexibility of long chains induced by trans-vinylene groups. Nevertheless, within the 2.5 A resolution available for direct lattice images (reported elsewhere), there is virtually no detectable structural change at the 10 e A-2 electron dose used for these exposures.

Improvements in technique have produced electron micrographs of purple membrane that provide, after computer analysis, reproducibly measurable diffraction peaks extending to 2.8 A (1 A = 0.1 nm). The improvements include better specimen preparation, a more stable cryo-electron microscope with better alignment and the addition of an image-processing step, which gives weights to local areas of the image according to the local strength of the periodic component of the image. These improvements have enabled the calculation of a directly phased projection map at 2.8 A resolution.

Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli. Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane. In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens. The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability. Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A. Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A. In the final projection maps, amplitudes from electron diffraction and phases from these images were combined. The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer. Since the molecule consists of almost exclusively β-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical β-pleated sheet crossing the membrane nearly perpendicularly to its plane. Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.

A simple procedure is shown for measuring the phase shift due to axial coma by superposing two micrographs with different axial coma in a laser diffractometer.

Electron micrographs of the purple membrane have been recorded using liquid nitrogen and liquid helium cooling on three cryoelectron microscopes. The best micrographs show optical diffraction spots, arising from the two-dimensional crystal, out to resolutions of around 6 A. Large areas of several of these micrographs have been analysed using a procedure which determines the strength of the very weak high resolution Fourier components of the image of the crystal. The procedure consists of reciprocal space filtering followed by real space correlation analysis to characterise image distortions, removal of the distortions by interpolation, and finally extraction of the amplitudes and phases of the Fourier components from the distortion-corrected image of the crystal. These raw image amplitudes and phases are then used, together with previously measured amplitude and phase information, to refine the beam tilt and crystal tilt, phase origin and amount of defocus and astigmatism of the image. The phases can then be corrected for the effects of the contrast transfer function, beam tilt and phase origin. The amplitudes of all the spots which are expected to be strong from their known electron diffraction intensity are observed to be significantly above the background noise level, and the independent phases from different images, and from symmetry-related directions in the same image, show excellent agreement out to a resolution of 3.5 A. Although only images from untilted or slightly tilted (

The advantage of MgO platelet crystals as specimen supports is shown. The contrast transfer of carbon foil granulation through the thin MgO platelet crystals was measured. The reduction of the contrast due to the crystal of 40 nm thickness was about 50%. Gold particles and ferritin particles were used as test specimens on MgO platelet crystals. The lattice images of both specimens were observed through the crystals. The protein shell of ferritin was recognizable. Some different problems arising with this specimen support are discussed.

Crotoxin complex forms thin crystals which are suitable for electron crystallographic analysis. We have used a 100 kV electron microscope equipped with a superconducting lens to image this crystal embedded in glucose. Optical diffraction analysis of the micrographs show unambiguously a structural resolution of 3.9 A which has not been obtained with the conventional microscope at room temperature. A density map with a nominal resolution of 3.5 A has been synthesized from these images by computer processing techniques.

Phase-contrast noise inherent in high resolution bright field images can be reduced considerably by using hollow-cone illumination instead of the usual axial illumination. It is shown by experiment that there are definite values of hollow-cone angle and defocus for imaging heavy-atom structures on an amorphous carbon film with minimum disturbing noise. The imaging is distinguished by a point-spread function whose side lobes extend over only a short range. Thus the present results may be considered as apodization in phase-contrast electron microscopy. The method is advantageous in investigating atomic structures where phase-contrast noise is a limiting factor for the visualization of high resolution details.

The effect of radiation exposure on visual recognition of individual macromolecules of the protein protamine was examined using high resolution electron microscopy in scanning transmission, fixed beam transmission, dark field and bright field, and recording of images on plates or via video imaging systems including image intensifiers and digital image storage. Loss of recognition of protamine, including its 5 A substructure, followed approximately three-hit kinetics with a D 0 on the exponential portion of the curve of about 470 e/A 2 . In spite of the inevitable chemical damage at high doses, virtually all molecules in orientations that exhibited the characteristic protamine structure could still be recognized in dark field scanning transmission or fixed beam bright field at 100–200 e/A 2 . Recognition fell from 30 to 6% when doses were increased from 1000 to 2000 e/A 2 in fixed beam dark field. Exposure rates, varied over seven orders of magnitude from 0.15 e/A 2 -s to 3 × 10 e/A 2 -s, had no effect at all on the recognition of structures.

Alignment by means of current commutating and defocusing of the objective does not yield the desired rotational symmetry of the imaging pencils. This was found while aligning a transmission electron microscope with a single field condenser objective. A series of optical diffractograms of micrographs taken under the same tilted illumination yet under various azimuths have been arranged in a tableau, wherein strong asymmetry is exhibited. Quantitative evaluation yields the most important asymmetric aberration to be the axial coma, which becomes critical when a resolution better than 5 A is obtained. The tableau also allows an assessment of the three-fold astigmatism. A procedure has been developed which aligns the microscope onto the coma-free and dispersion-free pencil axis and does not rely on current commutation. The procedure demand equal appearance of astigmatic carbon film images produced under the same tilt yet diametrical azimuth.

Calculated defect diffuse scattering patterns, using the kink defect model employed earlier to explain changes in Bragg electron diffraction intensities for heated n-paraffins, are shown to account best for observed diffuse data from solution-grown and epitaxially grown micro-crystals. The presence of significant diffuse scattering below 15°K, moreover, indicates that such chain kinks are stable in the crystal lattice.

Cross-correlation of micrographs taken under identical optical conditions allows measurement of (a) the optical transfer function free from electron and photographic noise and (b) object change, especially that due to radiation damage. Light optical methods based on superposition diffractograms are discussed. A subtraction procedure is described for measuring the optical transfer function, and a two-step diffraction process is presented for measuring object change. As an example, experiments with radiation damage in carbon foil are described. Here increased stability regarding radiation-induced change was found with increasing dose, making possible measurement of deviations from the “zero dose image”.

Background: The ribosome — essential for protein synthesis in all organisms — has been an evasive target for structural studies. The best available structures for the 70S Escherichia coli ribosome or its 30S and 50S subunits are based on electron microscopical tilt experiments and are limited in resolution to 28–55 a. The angular reconstitution approach, which exploits the random orientations of particles within a vitreous ice matrix, can be used in conjunction with cryo-electron microscopy to yield a higher-resolution structure. Results Our 23 a resolution map of the 70S ribosome elucidates many structural details, such as an extensive system of channels within the 50S subunit and an inter-subunit gap ideally shaped to accommodate two transfer RNA molecules. The resolution achieved is sufficient to allow the preliminary fitting of double-helical regions of an earlier three-dimensional ribosomal RNA model. Conclusion Although we are still a long way from attaining an atomic-resolution structure of the ribosome, cryo-electron microscopy, in combination with angular reconstitution, is likely to yield three-dimensional maps with gradually increasing resolution. As exemplified by our current 23 a reconstruction, these maps will lead to progressive refinement of models of the ribosomal RNA.